This application is focused on studying human T cells during the induction of type 1diabetes (T1D) and the breakdown of immunological tolerance. We seek to develop and utilize a novel humanized mouse model system to directly assess clinically relevant therapies to induce T cell tolerance and cure diabetes. We have developed a human relevant mouse model that we can imagine in vivo, in real time to address the basic mechanisms underlying the interplay between the immune cells and the target pancreatic beta cells during diabetes. This model system will help facilitate the testing of therapeutics to selectively target diabetogenic human T cells for treatment of T1D, as a pre-clinical model and a bridge between bench and bedside. The overall objective of this application is to utilize humanized mouse models to identify the major immune targets responsible for diabetes and selectively silence only these destructive T cells. Our central hypothesis is that islet antigens, including insulin, preproinsulin, GAD, IA2, and IGRP are major targets of the immune system and lead to beta cell death and diabetes. Our hypothesis has been formulated on the basis of strong research demonstrating that insulin protein chemically fixed to tolerogenic cells was able to reverse diabetes in newly diabetic NOD mice. Despite the role of these proteins as targets for diabetes pathogenesis in mouse models, we do not fully understand the specific T cell responses in human patients suffering from T1D. We have initiated a strong collaboration to investigate these immune targets using humanized mouse models to interrogate human immune cells during diabetes development. Using a transgenic mouse expressing the diabetes linked HLA I and HLA II alleles for HLA-A2.1 and HLA-DQ8 on the NOD.scid.IL-2-Rgamma c-/- mouse MHC I-/- MHC II-/- background we will test tolerogenic therapies to selectively target these diabetes relevant proteins as a translational T1D cure. The goals of this application are to 1) Establish and validate a human HLA humanized mouse model capable of multi-lineage immune reconstitution and diabetes development from HLA matched diabetic patients, 2) Determine the dynamic motility and cellular interactions of human T cells with islet graft tissue during the breakdown of tolerance and induction of diabetes in the humanized mouse using intravital two-photon imaging, and 3) Characterize and determine the functional and biological immune response against transplanted islets following antigen specific tolerance induction in the humanized model for long term islet graft survival.